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  • 學位論文

金奈米粒子於血漿免疫蛋白和生物硫醇分子之感測

Gold Nanoparticles for Sensing of Plasma Immunoproteins and Biological Thiols

指導教授 : 張煥宗
共同指導教授 : 黃志清

摘要


我們研發兩種偵測生物分子的技術。首先我們利用11-巰基十一酸(11-mercaptoundecanoic acid,11-MUA)保護的螢光金量子點(gold nanodot,Au ND)偵測血漿中免疫球蛋白G(immunoglobulin G,IgG)。IgG為人體血漿中含量最多的抗體,其正常濃度範圍在34.0–105 μM。血漿IgG不足時會造成免疫力下降,過量時則為類風濕性關節炎及紅斑性狼瘡等自體免疫疾病的生理指標。藉由蛋白質A(protein A)與IgG之間良好的親和力,當修飾protein A的Au ND(PA-Au ND)與不同濃度的IgG反應時,PA-Au ND的螢光會隨IgG濃度增加而增加,因此PA-Au ND可做為血漿中IgG的探針,其偵測極限可達10 nM,線性範圍在50–250 nM(R2 = 0.998)。此方法對IgG的選擇性高─與血漿運鐵蛋白(transferrin)相較選擇性達117倍。將此方法應用於2位健康成人血漿IgG的偵測,所測得的濃度為45.4 ± 3.2及58.0 ± 4.7 μM。   其次,我們應用修飾螢光染料分子尼羅紅(Nile Red,NR)的金奈米粒子(gold nanoparticle,Au NP)來選擇性的偵測單一種的生物硫醇分子(biological thiol)。當Au NP表面吸附NR時,兩者間會產生螢光共振能量轉移(fluorescence resonance energy transfer,FRET)及電子轉移(electron transfer),導致NR受Au NP消光。將生物硫醇分子加至NR-Au NP溶液時,其間會因形成強的硫金鍵結,而將NR從Au NP表面取代出來,導致溶液的螢光大幅增加。藉由觀察螢光改變程度,我們分別研究cysteine(Cys)、homocysteine(Hcy)和glutathione(GSH)的取代動力學,結果顯示取代速率順序為:Cys ~ Hcy > GSH。由於Cys溶液(pH 7.00)在空氣中加熱至95 ℃較Hcy和GSH易氧化,加熱後Cys溶液的取代動力學較Cys和Hcy緩慢4倍。藉由測量加熱1小時前後Cys溶液取代的螢光差值,可偵測Cys;其偵測極限達10 nM,線性範圍在100–1000 nM(R2 = 0.995)。此方法比直接取代NR-Au NP的結果(LOD 15 nM)更為靈敏。為提高此方法對Cys的選擇性,我們使用聚乙烯吡咯烷酮(polyvinylpyrrolidone,PVP)來修飾NR-Au NP─GSH取代PVP-Au NP表面NR較Hcy和GSH緩慢20倍。此方法目前已可應用於選擇性偵測血漿硫醇分子模擬溶液中的Cys,此硫醇分子模擬溶液中包含Cys(11.74 μM)、Hcy(0.301 μM)、GSH(3.511 μM)、半胱胺甘胺酸(cysteinylglycine,3.584 μM)及γ-麩胺醯胺半胱胺酸(γ-glutamylcysteine,0.566 μM)。兩次實驗所測得的Cys濃度為11.72 μM(回收率99.8%)及10.96 μM(回收率93.4%)。

並列摘要


We have developed two techniques for the detection of biomolecules of interest. First, 11-MUA-protected gold nanodots (Au NDs) have been prepared and employed for the detection of immunoglobulin G (IgG) in plasma. IgG is the most abundant antibody in plasma, with a normal concentration range over 34.0–105 μM. Plasma IgG at low levels causes poor immune systems, while at high levels can be indicators of autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Through the high-affinity binding between protein A and IgG, the fluorescence intensity of protein A-conjugated Au NDs (PA-Au NDs) increases upon increasing IgG concentration. For IgG, this approach provides a limit of detection (LOD) of 10 nM, a linear range over 50 and 250 nM (R2 = 0.998), and high selectivity (117-fold selectivity over transferrin). This approach has been validated by determining the concentrations of IgG in plasma samples from two healthy adults, with results of 45.4 ± 3.2 and 58.0 ± 4.7 μM (three measurements). Secondly, we have demonstrated the detection of biological thiols through their replacement of Nile Red (NR) from the surfaces of gold nanoparticles (Au NPs). When NR is adsorbed onto Au NPs, FRET and electron transfer occur, leading to fluorescence quenching. Upon addition of the biological thiols to the solutions of NR-Au NPs, NR molecules are released to the bulk solution, leading to increased fluorescence. By monitoring the fluorescence changes, we separately investigated the replacement kinetics of Cys, Hcy and GSH, showing the decreasing order Cys ~ Hcy > GSH. Since Cys in solution at pH 7.00 is oxidized in the air at 95 ℃ more rapidly than Hcy and GSH are, its replacement kinetic is significantly (4-fold) slower than that of the other two. By measuring the differences in fluorescence intensity of Cys solutions with and without being heated for 1 hr, this approach provides an LOD of 10 nM for Cys, with a linear range over 100–1000 nM (R2 = 0.995). This approach is more sensitive than that (LOD 15 nM) of direct replacement without heating. In order to improve the selectivity of this approach toward Cys, polyvinylpyrrolidone (PVP) had been used to modify NR-Au NPs. The replacement of NR from PVP-Au NPs by GSH is slower (20-fold) than those by Cys and Hcy, which allows selective detection of Cys in two prepared solutions composed of Cys (11.74 μM), Hcy (0.301 μM), GSH (3.511 μM), cysteinylglycine (3.584 μM) and γ-glutamylcysteine (0.566 μM), with results of 11.72 μM (recovery 99.8%) and 10.96 μM (recovery 93.4%).

參考文獻


第二章 修飾蛋白質A的螢光金量子點在免疫球蛋白G偵測的應用
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被引用紀錄


蔡香素(2013)。網版印刷碳電極表面修飾生物探針之初步探討〔碩士論文,中山醫學大學〕。華藝線上圖書館。https://doi.org/10.6834%2fCSMU.2013.00225

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